Kjell Madsen

Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes

Fetal guinea-pig epiphyseal chondrocytes were cultured in monolayers and as aggregates in the presence of antimicrotubular agents. Colchicine and vinblastine caused a dissociation of the Golgi complex, in addition to the disappearance of microtubules. Synthesis and secretion of proteoglycans and collagen were studied using radioactive precursors. Colchicine inhibited the synthesis of proteoglycans. The drug also inhibited secretion with an intracellular accumulation of these molecules. The proteoglycans secreted by the colchicine-treated cells had a smaller molecular size and contained a smaller proportion of aggregated molecules than proteoglycans in control cultures. However, there was no difference in the average size of the chondroitin sulfate side chains of the proteoglycan molecules. Nor was there any increase in the breakdown of proteoglycans in colchicine-treated cultures. Vinblastine was also found to inhibit synthesis and secretion of proteoglycans. Deuterium oxide also inhibited the synthesis of these molecules but stimulated their secretion into the medium. Colchicine caused an inhibition of both synthesis and secretion of collagen. It is suggested that the quantitative and qualitative effects of colchicine could be the result of disturbances in the Golgi complex, possibly in combination with a retarded translocation of secretory vacuoles. However, as the colchicine-treated chondrocytes were still able to continue a large part of their matrix biosynthesis with only moderate changes in the structure of the secreted molecules, it is probable that alternative pathways for the secretion of matrix molecules exist and/or the Golgi complex is able to retain a major part of its function despite the structural alterations.

Secretion of proteoglycans by chondrocytes: Influence of colchicine, cytochalasin B, and β-d-xyloside

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.

Production of cartilage-typic proteoglycans in cultures of chondrocytes from elastic cartilage

Abstract Chondrocytes from rabbit ear cartilage were isolated and cultured as monolayers in Hams F-12 medium. The proteoglycans synthesized by short-term cultures formed a high proportion of aggregates and contained chrondroitin-4- and -6-sulfate in a 2:1 proportion. Dermatan sulfate was not present. The average molecular weight of the chondroitin sulfate was about 20,000. Keratan sulfate with an average molecular weight of about 6000 could be isolated from the proteoglycan monomers. Rabbit ear chondrocytes in culture thus produced proteoglycans comparable to those isolated from hyaline cartilage. Culture for longer periods and plating at lower density caused a decrease in the proportion of aggregated proteoglycans. Primary cultures continued to synthesize aggregated proteoglycans for at least 2 weeks, while subdivision of the cultures caused a shift toward the production of small-sized “ubiquitous proteoglycans.” The synthesis of proteoglycan aggregates could, however, be partly restored by transfer of the monolayer cells to a suspension culture.

Cell fractions from rat rib growth cartilage. Biochemical characterization of matrix molecules.

In an attempt to isolate and characterize the putative target cells for growth hormone, chondrocytes were isolated from rat rib growth cartilage and fractionated by centrifugation in a discontinuous Percoll gradient. This resulted in three cell fractions with differing properties. The fraction with the lowest density consisted mainly of large, lipid-containing cells which became flattened in subsequent culture. The cells in this fraction were fair collagen producers but synthesized only minor amounts of proteoglycans and apparently no proteoglycan aggregates. These cells probably originate in the hypertrophy zone of the growth plate. The fraction with highest density, on the other hand, consisted of small cells which upon cell culture became polygonal and surrounded with refractile matrix. They synthesized less collagen, but more proteoglycans than the low-density fraction. The proportion of proteoglycan aggregates was rather low (10-20%) but otherwise the proteoglycans were similar to those synthesized by other chondrocytes. This cell fraction was tentatively identified as cells from the upper part of the growth plate. Finally, the middle fraction was heterogeneous, consisting of cells of varying shape. This fraction synthesized large amounts of both collagen and proteoglycans. In all three cell fractions, type II collagen predominated. There were also minor amounts of (1a, 2a, 3a) collagen, and, in the two denser fractions, of type I collagen.

Synthesis and secretion of proteoglycans by cultured chondrocytes: Effects of monensin, colchicine and β-d-xyloside☆

Abstract Chondrocytes, isolated from elastic ear cartilage of young rabbits, were grown in monolayer cultures in Hams F-12 medium. Synthesis and secretion of macromolecules were monitored by labelling with radioactive precursors and the effect of monensin and other experimental agents was investigated. Monensin caused an inhibition of the incorporation of precursors into macromolecular material and a moderate intracellular accumulation when used in higher concentrations. The effect was more pronounced for 35 SO 4 than for 3 H-labelled glucose or proline. p -Nitrophenyl-β- d -xyloside alleviated this inhibition to some extent, but there was a concomitant increase in the amount of intracellular labelled material. Colchicine and monensin together caused a severe inhibition of the incorporation of 35 SO 4 and a marked shift of the label to the intracellular compartment. Colchicine also increased the sensitivity of the cells to monensin, lowering the minimal effective concentration about one order of magnitude. The latter results are consistent with the idea that cytoplasmic microtubules have a stabilizing function on the secretory pathways and, that their removal by colchicine, causing a ‘randomizing’ of the Golgi complex, makes these pathways more vulnerable to monensin.

Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function.

Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.

Proteoglycans synthesized by fetal Guinea pig chondrocytes in culture

Short term cultures were carried out with chondrocytes and tissue fragments from fetal guinea pig epiphyseal cartilage. Proteoglycans were isolated from these cultures and their properties were compared with those of proteoglycans from adult hyaline cartilage. It was concluded that the proteoglycans synthesized in culture were essentially similar to those present in cartilage matrix in vivo. The authors therefore suggest that fetal guinea pig chondrocytes cultured in monolayer or as aggregates in suspension constitute a useful system for the study of synthesis and secretion of proteoglycans.

Direct Action of Growth Hormone on Cartilage Growth

It is generally considered that the stimulatory effect of growth hormone (GH) on skeletal growth and development is mediated by GH-dependent plasma factors—somatomedin(s)—that are produced in the liver in response to GH.(1,2) This theory is mainly based on the fact that it has been difficult to demonstrate stimulatory effects of GH in vitro on cartilage metabolism, whereas somatomedin(s), when added in vitro, stimulate a number of metabolic and cellular processes in cartilage tissue.(1,2) Recently, Schoenle et al. (3) demonstrated that continuous administration of highly purified IGF-I (somatomedin C) to hypo-physectomized rats, by means of osmotic minipumps, stimulated epiphyseal cartilage growth, supporting this hypothesis. The effect observed was comparable to that of small doses of continuously infused GH (12.5–25 mU hGH/day).

Glycosaminoglycan secretion from perifused monolayer cultures of rabbit ear chondrocytes: Modification of xyloside effect by colchicine and cytochalasin B

Abstract Chondrocytes isolated from elastic ear cartilage of young rabbits were grown in monolayer cultures. Secretion into the medium of glycosaminoglycans labeled with 35SO4 was monitored by continuous perifusion. This experimental system was used to study the influence of colchicine, lumicolchicine, and cytochalasin B on the response to a 2-h β- d -xyloside pulse. In untreated cultures the stimulative effect of the xyloside was seen within 6 min and a new secretory steady state was reached in less than 20 min. Rabbit ear chondrocytes thus respond considerably faster to β- d -xyloside than chick sternum chondrocytes. After xyloside withdrawal glycosaminoglycan production decreased to baseline amounts in about 30 min. Colchicine caused a delayed and protracted response to xyloside. These effects were not mimicked by lumicolchicine. Hence, cytoplasmic microtubules appear to be involved in the secretion of glycosaminoglycans. On the other hand, colchicine had no effect on the size distribution of the labeled macromolecules. These findings suggest that colchicine exerts its action at a stage subsequent to initiation of the synthesis of glycosaminoglycans. Cytochalasin B did not affect the rate of response to xylside exposure or withdrawal.